Plasmids are commonly used to multiply (make many copies of) or express particular genes.

LEARNING OBJECTIVES

Recognize the characteristics of, and thus the functions, of plasmids

KEY TAKEAWAYS

Key Points

The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics, and into a multiple cloning site (MCS, or polylinker), allowing the easy insertion of DNA fragments.

A major use of plasmids is to make large amounts of proteins. Bacterium can be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.

It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions.

Key Terms

Col plasmids: These plasmids contain genes that code for bacteriocins, proteins that can kill other bacteria.

F-plasmid: Fertility F-plasmids contain tra genes and are capable of conjugation resulting in the expression of sex pilli.

Resistance plasmids: These plasmids contain genes that provide resistance against antibiotics or poisons.

Types of Plasmids

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics. The gene is also inserted into a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments.

A Plasmid Map of pUC19: pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the multiple cloning site region is reversed.

Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter, selecting only the modified bacteria. Finally, these bacteria can be grown in large amounts, harvested, and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.

One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA, and may possibly be a tool for curing many diseases.

It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Thus, plasmids can be assigned into incompatibility groups.

Another way to classify plasmids is by function. There are five main classes:

Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli.

Resistance plasmids, which contain genes that provide resistance against antibiotics or poisons. They were historically known as R-factors, before the nature of plasmids was understood.

Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.

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